Rose Y. Reins

Antibacterial activity and cytotoxicity of PEGylated poly(amidoamine) dendrimers

We have investigated the antibacterial activity and cytotoxicity of a series of amino-terminated poly(amidoamine) (PAMAM) dendrimers modified with poly(ethylene glycol) (PEG) groups. The antibacterial activity of the PAMAM dendrimers and their derivatives against the common ocular pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, was evaluated by their minimum inhibitory concentrations (MICs). For the unmodified third and fifth generation (G3 and G5) amino-terminated dendrimers, the MICs against both P. aeruginosa and S. aureus were in the range of 6.3-12.5 microg mL(-1), comparable to that of the antimicrobial peptide LL-37 (1.3-12.5 microg mL(-1)) and within the wide range of 0.047-128 microg mL(-1) for the fluoroquinolone antibiotics. PEGylation of the dendrimers decreased their antibacterial activities, especially for the Gram-positive bacteria (S. aureus). The reduction in potency is likely due to the decrease in the number of protonated amino groups and shielding of the positive charges by the PEG chains, thus decreasing the electrostatic interactions of the dendrimers with the negatively-charged bacterial surface. Interestingly, localization of a greater number of amino groups on G5 vs. G3 dendrimers did not improve the potency. Significantly, even a low degree of PEGylation, e.g. 6% with EG(11) on G3 dendrimer, greatly reduced the cytotoxicity towards human corneal epithelial cells while maintaining a high potency against P. aeruginosa. The cytotoxicity of the PEGylated dendrimers to host cells is much lower than that reported for antimicrobial peptides. Furthermore, the MICs of these dendrimers against P. aeruginosa are more than two orders of magnitude lower than other antimicrobial polymers reported to date. These results motivate further exploration of the potential of cationic dendrimers as a new class of antimicrobial agents that may be less likely to induce bacterial resistance than standard antibiotics.

Ocular Surface Expression and In Vitro Activity of Antimicrobial Peptides

Purpose: Human ocular surface epithelia express four antimicrobial peptides (APs): β -defensin (hBD) 1-3 and LL-37. Here the expression of additional APs (hBD 4-6, HE2β 1; histatin-1, -3; liver expressed antimicrobial peptide-1, -2; macrophage inflammatory protein (MIP)-3α, and thymosin (T)β -4) was sought and activity against common ocular pathogens studied. Methods: AP expression was determined in human corneal and conjunctival epithelial cells (HCEC, HCjEC) by RT-PCR and in corneal sections by immunostaining. Antimicrobial assays were performed to assess peptide (hBD 1-3, LL-37, MIP-3α, and Tβ 4) activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE) in the presence of NaCl or tears. Results: HCEC and HCjEC expressed MIP-3α and Tβ 4. hBD 1-3, MIP-3α, and Tβ 4 showed activity against PA. hBD-3 had potent activity against SA and SE, whereas hBD-2, MIP-3α and Tβ 4 had moderate activity and hBD-1 had none. NaCl markedly attenuated, and tears almost completely inhibited the activity of hBD 1-2 and Tβ 4, but not that of hBD-3. Conclusions: The ocular surface epithelia additionally express MIP-3α and Tβ 4 which have moderate antimicrobial activity. The current data support a role for hBD-3 as an antimicrobial peptide in vivo, but call in to question the effectiveness of some other APs. However, further study is required to conclusively elucidate the physiological role of each AP.

Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells.

The ability of the ocular surface to respond to pathogens is in part attributed to toll-like receptors (TLRs) that recognize conserved motifs on various microbes. This study examines TLR expression on various ocular surface cells, if TLR agonists can modulate the expression of antimicrobial peptides (AMPs), human beta defensins (hBD) and cathelicidin (hCAP-18/LL-37) which maybe functionally active against Pseudomonas aeruginosa (PA) and if TLR agonists or AMPs can modulate TLR mRNA expression. TLR1-10 mRNA expression was examined in corneal epithelial, corneal stromal cells and conjunctival epithelial cells by RT-PCR. To confirm protein expression flow cytometry or immunostaining was performed for selected TLRs on some cell cultures. Ocular surface cells were cultured with a range of TLR agonists and then hBD-1, 2, 3, or hCAP-18 mRNA and protein expression was determined by RT-PCR and immunoblotting. In some experiments, cells were cultured with a cocktail of agonists for TLR3, 5 and 6/2 and the antimicrobial activity of the culture media was tested against PA. TLR mRNA expression was also examined in primary human corneal epithelial cells (HCEC) treated with either 3 μg/ml of hBD-2, 5 μg/ml of LL-37 or TLR4, 5 and 9 agonists. Overall, the ocular surface cells expressed mRNA for most of the TLRs but some differences were found. TLR2 was not detected in corneal fibroblasts, TLR4 was not detected in primary cultured or freshly isolated HCEC, TLR5 was not detected in conjunctival epithelial cells (IOBA-NHC) and corneal fibroblasts, TLR7 was not detected in freshly isolated HCEC and TLR10 was not detected in HCEC and IOBA-NHC. TLR8 mRNA was not expressed by any of the samples tested. Immunostaining of cadaver corneas revealed TLR5 and 9 expression throughout the cornea while TLR3 was significantly expressed only in the epithelium. Flow cytometry and immunostaining revealed cultured fibroblasts expressed TLR9 but had no significant TLR3 expression. hBD-2 expression was upregulated by TLR1/2, 3, 4, 5 and 6/2 agonists depending on the cell type, whereas only the TLR3 agonist upregulated the expression of hCAP-18 in primary HCEC. The combination of TLR3, 5 and 6/2 agonists in primary HCEC, upregulated hBD-2 and hCAP-18 mRNA and peptide expression and secretion into the culture media, which significantly killed PA. This antimicrobial activity was primarily attributed to LL-37. TLR agonists did not modulate TLR expression itself, however, LL-37 or hBD-2 downregulated TLR5, 7 and/or 9 mRNA depending on the cell type. TLRs are expressed on the ocular surface and TLR agonists trigger the production of LL-37 and hBD-2, with LL-37 being particularly important for protecting the ocular surface against PA infection.

In vitro activity of human β-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid

ABSTRACT Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human β-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

Vitamin D Activation and Function in Human Corneal Epithelial Cells During TLR-Induced Inflammation.

PURPOSE Vitamin D is recognized to be an important modulator of the immune system. In the eye, studies have shown that deficiencies and genetic differences in vitamin D-related genes have a significant impact on the development of various ocular diseases. Our current study examines the ability of human corneal epithelial cells (HCEC) to activate vitamin D and the effect of vitamin D treatment on antimicrobial peptide production and cytokine modulation during inflammation, with the ultimate goal of using vitamin D therapeutically for corneal inflammation. METHODS Human corneal epithelial cells were treated with 10-7M vitamin D3 (D3) or 25-hydroxyvitamin D3 (25D3) for 24 hours and 1,25-dihydroxyvitamin D3 (1,25D3) detected by immunoassay. Human cathelicidin (LL-37) expression was examined by RT-PCR, immunoblot, and immunostaining following 1,25D3 treatment and antimicrobial activity of 1,25D3-treated cells was determined. Cells were stimulated with TLR3 agonist polyinosinic-polycytidylic acid (Poly[I:C]) for 24 hours and cytokine levels measured by RT-PCR, ELISA, and Luminex. Immunostaining determined expression of vitamin D receptor (VDR) and retinoic acid inducible gene-1 receptor (RIG-1) as well as NF-κB nuclear translocation. RESULTS When treated with inactive vitamin D metabolites, HCEC produced active 1,25D3, leading to enhanced expression of the antimicrobial peptide, LL-37, dependent on VDR. 1,25-D3 decreased the expression of proinflammatory cytokines (IL-1β, IL-6, TNFα, and CCL20) and MMP-9 induced by Poly(I:C) as well as pattern recognition receptor expression (TLR3, RIG-1, MDA5). However, early activation of NF-κB was not affected. CONCLUSIONS These studies demonstrate the protective ability of vitamin D to attenuate proinflammatory mediators while increasing antimicrobial peptides and antipseudomonas activity in corneal cells, and further our knowledge on the immunomodulatory functions of the hormone.

Vitamin D: Implications for Ocular Disease and Therapeutic Potential

Vitamin D is a multifunctional hormone that is now known to play a significant role in a variety of biological functions in addition to its traditional role in regulating calcium homeostasis. There are a large number of studies demonstrating that adequate vitamin D levels are important in maintaining health and show that vitamin D is able to be utilized at local tissue sites. In the eye, we have increasing evidence of the association between disease and vitamin D. In this narrative review, we summarize recent findings on vitamin D and its relationship to various ocular pathologies and the therapeutic potential for some of these, as well as examine the basic science studies that demonstrate that vitamin D is biologically relevant in the eye.

Effects of Topically Applied Vitamin D during Corneal Wound Healing

Vitamin D is an important regulator of immune function and largely acts to dampen chronic inflammatory events in a variety of tissues. There is also accumulating evidence that vitamin D acts to enhance initial inflammation, beneficial during both infection and wound healing, and then promotes resolution and prevention of chronic, damaging inflammation. The current study examines the effect of topical vitamin D in a mouse of model of corneal epithelial wound healing, where acute inflammation is necessary for efficient wound closure. At 12 and 18 hours post-wounding, vitamin D treatment significantly delayed wound closure by ~17% and increased infiltration of neutrophils into the central cornea. Basal epithelial cell division, corneal nerve density, and levels of VEGF, TGFβ, IL-1β, and TNFα were unchanged. However, vitamin D increased the production of the anti-microbial peptide CRAMP 12 hours after wounding. These data suggest a possible role for vitamin D in modulating corneal wound healing and have important implications for therapeutic use of vitamin D at the ocular surface.

MyD88 contribution to ocular surface homeostasis

The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88-/- tissues compared to WT and IL-1R-/- mice. Interestingly, MyD88-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm2) than both WT (0.59±0.16 gm/mm2) and IL-1R-/- (0.52±0.08 gm/mm2). Following Pseudomonas aeruginosa challenge, MyD88-/- mice had better clinical scores (0.5±0.0) compared to IL-1R-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.

Vitamin D Induces Global Gene Transcription in Human Corneal Epithelial Cells: Implications for Corneal Inflammation

Purpose Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we further examined the timing of the cytokine response to combined vitamin D–TLR treatment and used genome-wide microarray analysis to examine the effect of vitamin D on corneal gene expression. Methods Telomerase-immortalized HCEC (hTCEpi) were stimulated with polyinosinic-polycytidylic acid (poly[I:C]) and 1,25-dihydroxyvitamin D3 (1,25D3) for 2 to 24 hours and interleukin (IL)-8 expression was examined by quantitative (q)PCR and ELISA. Telomerase-immortalized HCEC and SV40-HCEC were treated with 1,25D3 and used in genome-wide microarray analysis. Expression of target genes was validated using qPCR in both cell lines and primary HCEC. For confirmation of IκBα protein, hTCEpi were treated with 1,25D3 for 24 hours and cell lysates used in an ELISA. Results Treatment with 1,25D3 increased poly(I:C)-induced IL-8 mRNA and protein expression after 2 to 6 hours. However, when cells were pretreated with 1,25D3 for 24 hours, 1,25D3 decreased cytokine expression. For microarray analysis, 308 genes were differentially expressed by 1,25D3 treatment in hTCEpi, and 69 genes in SV40s. Quantitative (q)PCR confirmed the vitamin D–mediated upregulation of target genes, including nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα). In addition to increased transcript levels, IκBα protein was increased by 28% following 24 hours of vitamin D treatment. Conclusions Microarray analysis demonstrates that vitamin D regulates numerous genes in HCEC and influences TLR signaling through upregulation of IκBα. These findings are important in dissecting the role of vitamin D at the ocular surface and highlight the need for further research into the functions of vitamin D and its influence on corneal gene expression.

High-Mobility Group Box 1 in Dry Eye Inflammation

Purpose To determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation. Methods EDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining. Results EDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment. Conclusions HMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.